Novel methods of inhibiting the activity of testosterone 5{60 -reductase

ABSTRACT

A novel method of inhibiting or arresting the androgenic manifestations of testosterone and other androgens herein disclosed. In this novel method, the conversion of testosterone into its active form, namely 5 Alpha -dihydrotestosterone by the action of the enzyme testosterone 5 Alpha -reductase is impaired by the introduction into the metabolic system of certain compounds which inhibit the action of the enzyme. The compounds which inhibit the action of this enzyme also inhibit the growth of sebaceous organs.

United States Patent Voigt et al.

[ Nov. 4, 1975 NOVEL METHODS OF INHIBITING THE ACTIVITY OF TESTOSTERONESa-REDUCTASE Inventors: Walter Voigt; Sung Lan Hsia, both of Miami, Fla.

Assignee: Research Corporation, New York,

Filed: Aug. 20, 1973 Appl. No.: 389,741

Related US. Application Data Continuation-impart of Ser. No. 201,592,Nov. 8,

Foreign Application Priority Data Nov. 8, 1972 Canada 156016 US. Cl.424/242: 424/243 Int. Cl. A61K 31/56 Field of Search 424/242, 238;260/397.4,

References Cited OTHER PUBLICATIONS Steroids (1969) by Nayfeh et al.,Vol. 14, No. 3, pp. 269-283.

J. Biol. Chem. (1973), No. 12, by Voight et al., pp. 4280-4285.

Primary ExaminerElbert L. Roberts Attorney, Agent, or FirmOmri M. Behr[57] ABSTRACT pounds which inhibit the action of this enzyme alsoinhibit the growth of sebaceous organs.

6 Claims, No Drawings NOVEL METHODS OF INHIBITING THE ACTIVITY OFTESTOSTERONE a REDUCTASE RELATED APPLICATIONS This application is acontinuation-in-part of our copending application Ser. No. 201,592,filed November 8, 1971, and also claims partial priority from Canadianapplication Ser. No. 156016, filed November 8, 1972, under licenses No.362646 dated October 30, 1972 and 362750 dated November 2, 1972.

FIELD OF THE INVENTION Novel anti-androgenic and anti-seborrheicprocedures.

DESCRIPTION OF THE PRIOR ART It is well known in the hormonal arts thatcertain undesirable physiological manifestations are caused by anexcessive accumulation of testosterone or similar androgenic hormones inthe metabolic system, in particular, in the male system. One of theseundesirable manifestations is the excessive enlargement of the prostategland which is fairly common in males of age 60 or over. Compounds whichcounteract the action of androgens are well known in the art. However,many of these compounds, for example, the estrogens, not only counteractthe effect of the androgens but have a feminizing effect as well. Thisfeminizing effect is of course totally undesirable and has held back thetreatment of conditions caused by excess androgenic hormones bychemotherapeutic means. Heretofore it has been believed thattestosterone, or a metabolite thereof is bound to the prostate glandcausing excessive growth thereof. The anti-androgenic compounds knownand used heretofore had as their modus operandi the prevention of thebinding of the testosterone to the gland, and as to the pituitary.

It has been strongly postulated heretofore, that testosterone isconverted by an enzyme in target tissues into Sa-dihydrotestosterone.Sa-dihydrotestosterone is a far more potent androgen than testosteroneitself and it is believed that it is this reduced hormone (hereinafterSa-DHT which is actually responsible for the dysfunction of the prostategland. Hence, if a mode could be found to inhibit the conversion oftestosterone into Sa-DHT then the dilemma posed by the use of feminizinghormones as anti-androgens might be eliminated.

SUMMARY OF THE INVENTION The invention described herein was made in thecourse of work under a grant or award from the Department of Health,Education and Welfare. We have made the surprising discovery thatcertain substantially unsubstituted A4-3-keto-androstanes substituted bycertain B-oriented substituents at C inhibit the action of testosterone5a-reductase. More specifically the 17B-substituent should either be anoxygen bonded to a carboxycarbon or have an oxygen attached to the Ccarbon. Furthermore, the a-position at C should be unsubstituted. Fluorosubstitution at C -oz has also been found helpful.

We advance no theory whatsoever as to the reasons why compounds havingthese structural characteristics inhibit the action of testosteroneSa-reductase in androgen sensitive organs. We merely note that whilecompounds having similar structure to this group include progesterone acompound convertible in vivo to androgens and androstenedione which is aknown androgen, have testosterone Sa-reductase inhibiting activity, thecompounds utilized in the present invention do not possess the undesiredandrogenic properties of these named compounds, nor the feminizingeffect of estrogens. It has further been observed that the foregoingcompounds are useful as growth inhibitors of seba- CCOUS organs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The A4-3-keto androstanes ofthe present invention which act as testosterone Sa-reductase inhibitorsmay carry certain l7B-oxy groups or may carry oxygen containing groupsatC having a ,B-orientation.

These include. haloalkanoyloxy groups, for example, fluoroacetoxy,bromoacetoxy or iodoacetoxy, hydroxy alkanoyl substituents such as thehydroxy acetyl groups, and alkanoyl derivatives thereof suitably acetoxyalkanoyl' groups such as acetoxy acetyl, and most desirable of all, isthe carboxylic acid group. It has further been found that the activityof certain of these active compoundsmay be further enhanced by thesubstitution of a l6-a fluoro atom.

The preferred compounds of the present invention may be structurallyrepresented as follows:

wherein R is selected from the group consisting of COOH, COOR where R isalkyl, or phenyl alkyl, 0R is haloalkyl, suitably chloromethyl oriodomethyl, and R, is hydroxy or alkanoyloxy, suitably acetoxy. In theforegoing definition, the alk moiety is preferably lower alk, suitablyhaving l-5 carbon atoms. R is hydrogen or fluorme.

In contrast to the foregoing it should be noted the inhibition oftestosterone Sa-reductase in androgen sensitive organs such as theprostate gland, the seminal vesicles, and the sebaceous glands isdecreased when a substituent ispresent at 17-a and is substantiallydecreased when no oxygen is present in the C -B substituent in themanner mentioned hereinabove. The presence of an oxygen function at Cand the removal of the C methyl group as in the case of l9-Nortestoster-3 one, also decrease the inhibitory effect.

While we do not wish to bebound by the theory, it would appear that thefactors which inhibit the action of testosterone Sa-reductase' do so bycompetition for the Sa-reductase with testosterone. The foregoingfinding makes it possible to effect physiological administration tomammals requiring the same. Steroids falling within the aforementionedcategory of inhibiting testosterone a-reductase activity do so withoutexhibitingundesirable side-effects.

The compounds may be administeredin the usual manner to mammals namely,orally, parenterally, or intramuscularly. They may be formulated astablets, powders, or capsules, with the carriers and soliddiluents knownto the art, or they may be diluted with liquid diluents for injectionpurposes such as saline, sesame oil or the like.

It will be understood by those skilled in the art that the dosage amountand dosage frequency will depend upon the nature of the condition to becorrected. However, with that proviso, it maybe stated that there may beadministered from about 0.5 to about 5,. suitably from about 1 to about3 mg/kg. of the inhibiting steroid per dose and there may beadministered from about 1 to about suitably from about 2 to about 6mg/kg per day for from about to about 40 days of the inhibitor. Whilepreviousstudies have shown the Sa-reduction of progesterone by slices ofhuman skin it has not heretofore been found that a relationship existsbetween the Sa-reductase ,activity directed to progesterone and theSa-reductase activity directed to testosterone.

The compounds having the surprising Sa-reductase inhibiting activitydisclosed herein are, with the exception of the esters whose method ofsynthesis is disclosed herein below, old compounds which are well knownin the art or which may be produced by those skilled in the art bymethods which are well known to them.

It has further been noted that administration of these compoundssuppresses the growth of sebaceous organs in mammals. For this purposeit is preferred to administer these compounds topically. They may beformu lated as lotions, salves, creams or tinctures with the carriersand diluents known to the art.

Dosage amounts and dosage frequency will, it will be understood by thoseskilled in the art be varied in accordance with the nature of thecondition to be corrected. Nevertheless, there may be administereddosages of the order of 100 ug to 1000 pg] sq.cm/day suitably about200-400 ;tg/ sq.cm/day in dilutions of from about 1 mg. to about 20mg./ml. of diluent. The invention should not however be considered aslimited by these ranges.

EXPERIMENTAL Steroids Testosterone -4-C (specificactivity 58.8 #Ci perprnole was purchased from New England Nuclear. They showed only singlepeaks of radioactivityin our chromatography systems and were usedwithout further purification.

EXAMPLE 1 Homogenization of 4 Foreskin Samples Foreskin specimens wereobtained at circumcision of the newborn, and were either usedimmediatelyor stored at l5, "C. Specimens stored up to several monthswere found to suffer no loss of thetestosterone 5o -reductase activity.Approximately 1 g of tissue was first finely chopped with scissors andthen homogenized in 10 vol- 4 umes of a medium containing 0.1 Mphosphate and 0.2 M sucrose, pH 7.5, with a Polytron (Kinematica,Lucerne, Switzerland), until there were no more visible pieces of intactskin. Thisusually took less than 5 min. Bundles of collagen fibers whichresisted homogenization were removed with tweezers. The homogenate wasthen transferred into a Ten Broeck ho'mogenizer fitted with a U3horsepower motor operated at 1000 rpm and further ground by' 15 strokesof the pestle. The collage nous material was removed by filtrationthrough glasswool, and the filtrate was centrifuged at 800x g for 10min. The precipitate was washed three times with the homogenizingsolution and the pellet contained nuclei as the major component.Fractions designated mitochondrial and microsomal were obtainedfollowing centrifugation at 10,000 X g for 10 min and 105,000 X g for 1hour, respectively. They were resuspended in the homogenizing solution(mitochondrial pellet from 1 g of skin in 54 ml and microsomal pelletfrom 1 g of skin in 1 ml). All operations were carried out at 04.

EXAMPLE 1i Assay of Enzymatic Activity Unless otherwise indicated, skinfractions were incubated with testosterone-4-C (l0 dpm) dissolved in 20[.L] of ethanol and added to an incubation medium containing 5 umoles ofglucose 6-phosphate, 0.2 ,umole of NADP, and 0.2 unit of glucose6-phosphate dehydrogenase (one unit of glucose 6-phosphate dehydrogenaseis the amount of en-- zyme that reduces l umole of NADP in '1 min at pH7.4, 25 in the presence of glucose 6-phosphate) in a total volume of mlof 0.1 M citratephosphate buffer, pH 5.6. Incubations were carried outfor 10 min at 37 in a Dubnoff metabolic incubator.

The reaction was terminated by immersing the incubation flask in a DryIceacetone bath followed by lyophilization. Carrier steroids (50 ug eachof testosterone, Sa-dihydrotestosterone, and androstanediol) were addedand the mixture was extracted with methanol. The extract wasconcentrated under nitrogen, and the residue was applied to thin layerplates and chromatographed in a system of chloroform-methanol(98.25:-1.75) as described previously. The carrier steroids werevisualized by exposure to iodine vapor, and the radioactivity wasdetected with a Vanguard Autoscanner 880. The radioactive spotcorresponding to 5adihydrotestosterone were then scraped off the platesand radioassayed in a Packard Tri-Carb scintillation counter. Thescintillation fluid used was a mixture of 5 g of 2,5-diphenyloxazole(PPO), 300mg of dimethyl l ,4-bis[ 2-(5-phenyloxazolyl)] benzene (POPOP)(Packard), g of naphthalene, 240 ml of ethyl alco hol, 400 ml ofdioxane, and 400 ml of toluene. The amounts ofSa-dihydrotestosterone'produced was calculated from the C in thisfraction and the specific activity of the steroid (same astestosterone-4 KB). The testosterone Sa-reductase activity wascalculated from the sum of the two Sat-steroids. i

EXAMPLE lll Inhibitory effect of various steroids on Sa-reduction oftestosterone Aliquots ofa microsomal suspension (10 mg of protein) wereincubated with testosterone-4-C l0 dpm,

0.77 mumole) in the presence of the various nonradioactive steroids at afinal concentration of 10 v M. Incubation conditions are described underExample II.

Additions Inhibition 3oxol 7B-vinyl-androst-4-ene Testosteronel7B-chloroacetate Testosterone 173-7 iodo acetate EXAMPLE iv v Synthesisof Testosterone 17B-Chloroacetate 300 mg of testosterone were dissolvedin 60 ml of icecold acetone containing 0.6 ml of pyridine. 3.6 g ofchloroacetic anhydride in ml of acetone were added drop-wise over; aperiod of 30 min. The mixture was kept overnight at 4C and reduced to mlvolume under N ltrwas then added to 400 ml of ice-water. The precipitatewas .--collected, washed, and crystallized from acetone and water. Weobtained 352 mg of product.

Identification:

m.p. of testosterone 153 158 m.p. of product 125 mixed m.p. 115

IR spectrum run on testosterone and the expected chlo roacetate oftestosterone gave an ester peak at 1710 cm only on the chloroacetate.

EXAMPLE V Synthesis of Testosterone l7fi-lodoacetate 229 mg oftestosterone l7/3-chloroacetate were refluxed with 320 mg of K1 in 30 mlacetone for 4 hours. After the reaction, the volume was reduced to 5 ml.with N and the mixture added to ml. of an ice-cold 5% solution of Na S Oin water. The precipitate obtained was recrystallized fromacetonefandwater. Yield 254mg. Identification: m.p. of product 138. IR spectrum ofthe product showed also an esterpeak at 1710 cm. The rest of thespectrum had some features different than the spectrum of thetestosterone-chloroacetate.

In accordance with the foregoing procedure, but using potassium bromidein place of potassium iodide, there is obtained testosteronel7B-bromoacetate.

EXAMPLE VI The effect of certain compounds on a sebaceous gland (femalehamster flank organ) was tested by a modification of the method of Frostand Gomes (Advances in Biology of Skin. p.403, 12, Ed. Montagna et al.,Appleton-Century-Crofts, New York, New York, 1969). I

Materials and Methods Steroids Testosterone propionate was obtained fromMann Research: 4-Androstene-3-onel 7B-carboxylic acid.

The methyl ester of 4-androstene-3-one-l7B-carboxylic acid was preparedby methylation of the acid in late, 230 mg. was obtained afterprecipitation with the addition of water and recrystallization fromacetone and water, mp. 131C.

THE FLANK ORGAN TEST Female Syrian golden hamsters, ten weeks old, wereobtained from Engles Laboratory (Farmersburg, lndiana), and fed PurinaRat Chow ad libitum. At the beginning of treatment and every three daysthereafter, the animals were closely shaven with an electric hairclipper to expose the flank organ and adjacent skin for visualobservation. The animals were separated into three groups of 5animalseach and treated with the various steroids as follows. Group 1received testosterone propionate (4 pg); Group ll, testosteronepropionate (4 pg.) plus 4-androstene-3-one-17B-carboxylic acid (400 pg);Group lll, testosterone propionate (4 pg.) plus methyl4-androstene-3-one-l7B-carboxylate (400 pg). The steroid solution, in plof acetone, was applied topically to one of the flank organs of eachanimal, while 60 pl of acetone was applied to the contralateral organwhich was used as the control. A small pipette was used to deliver thesolution or acetone, and case was taken that the applied material didnot run from the area of the flank organ. One application was made daily(except Saturdays and Sundays) for three weeks. Exposed skin area ca. 2cm For histological studies, the organs were excised at the end oftreatment, cut in half and fixed in Carnoys fixative. Paraffin sectionswere cut through the center of the organ and stained in hematoxylin andeosin.

In animals of Group 1, following three weeks of applications oftestosterone propionate to one of the two organs, the treated'organincreased in size to resemble a male organ. The contralateral organ,however, remained small, indicating that the steroid at the dose usedexerted only local effect. This result is shown. In animals of Group 11treated concomitantly with testosterone propionate and4-androstene-3-one-l-7B-carboxylic acid, the stimulatory effect oftestosterone propionate was completely blocked, and at the end of threeweeks, the treated organ showed no increase in size or pigmentation, andwas indistinguishable from the contralateral control organ.

ln Group 111, it was found that methyl 4-androstene- 3-onel7B-carboxylate was equally inhibitory of the effects of testosteronepropionate as 4-androstene-3-onel7B-carboxylic acid.

Histological sections through the center of the flank organ also revealthe inhibitory properties of 4-androstene-3-one-l7B-carboxylic acid. Ina section of the flank organ of a female hamster stimulated withtestosterone propionate (Group I), enlarged sebaceous glands are notedwhich form an almost continuous mass from follicle to follicile. Asection through the organ of an animal treated with testosteronepropionate plus 4-androstene-3-one-17B-carboxylic acid (Group II)revealed small and widely separated sebaceous glands.

At greater magnification a majority of the individual sebaceous cells inthe stimulated organ exhibited a cytoplasm replete with lipid droplets.In contrast, in the 4-androstene-3-one-l7B-carboxylic acid treated flankorgan, the cells of the sebaceous glands contained much less cytoplasmiclipids. This observation suggests that the inhibitory effect of4-androstene-3-one-17B- carboxylic acid on sebaceous glands is not onlywith respect to the total number of sebaceous cells, but on their lipidcontent as well.

EXAMPLE VlI Androst-4-ene-3-one-l7B-carboxylic acid (referred to asAEOC) was further tested for its capacity to reduce the prostate glandand the seminal vesicles of rats.

For this test, Sprague-Dawley rats, 4 weeks old, were castrated anddivided into various groups of seven rats each. Starting the dayfollowing castration, treatment began in the manner described in Tablell. The daily injections of either testosterone propionate, AEOC, wereadministered subcutaneously with sesame oil as vehicle, and lasted forseven days. One day after the last injection, the rats were killed andthe ventral prostate and seminal visicles were excised and weighed.

It can be seen from the results that AEOC exerts an inhibitory effect onthe growth of both the prostate and the seminal vesicles of rats.

TABLE ll Dose Mean Organ Weight Gain Body Weight Group mg/rat/day mg. g.

T.P. AEOC v.1 s.v. F 0 24 31 46.7 A 0.08 0 96 133 57.4 D 0.08 8.0 53 7443.1 C 0.08 4.0 44 92 41.4 B 0.08 0.8 45 90 55.0 E 0 8.0 8 l6 41.9lntact Animals 0 50 43 49.2 Abbreviations: V.P.: ventral prostate S,V.:seminal vesicles T.P.: testosterone propionate AEOC: androst-4-ene-3-onel 713 -carboxylic acid We claim: 1. A method ofinhibiting the activity of testosterone -reductase in androgen sensitiveorgans of the group consisting of the prostate glands, the sebaceousglands and the seminal vesicals by administering to a mammal in need ofsuch inhibition, between 1 and mg/Kg/day of a compound of the fonnula:

wherein R is alkyl or phenyl alkyl, R is haloalkyl, 0R4 is hydroxy oralkanoyloxy, and R is hydrogen or fluoro, wherein alk signifies 1-5carbon atoms.

2. A method of claim 1 wherein in the compound of Formula 1, R is COOH.

3. A method of claim 2 wherein in the compound of Formula I is R, isCOOH or COOR 4. A method of claim 1 wherein the compound of Formula 1 Ris and OR, is hydroxy or alkoxy.

5. A method of claim 2 wherein the compound of Formula I R is -Q-CH ORand OR, is hydroxy or alkoxy.

6. A method of suppressing the growth of sebaceous organs in mammalswhich comprises administering thereto between 100 and 1000 ug/sq. cm/dayof:

wherein -R is selected from the group consisting of COOH, -COOR whereinR is alkyl or phenyl alkyl, R is alkyl or haloalkyl, OR, is hydroxy oralkanoyloxy, and R is hydrogen or fluoro, wherein alk signifies l-5carbon atoms.

1. A METHOD OF INHIBITING THE ACTIVITY OF TESTOSTERONE 5 -REDUCTASE INANDROGEN SENSITIVE ORGANS OF THE GROUP CONSISTING OF THE PROSTATEGLANDS, THE SEBACEOUS GLANDS AND THE SEMINAL VESICALS BY ADMINISTERINGTO A MAMMAL IN NEED OF SUCH INHIBITION, BETWEEN 1 AND 10 MG/KG/DAY OF ACOMPOUND OF THE FORMULA:
 2. A method of claim 1 wherein in the compoundof Formula I, R1 is -COOH.
 3. A method of claim 2 wherein in thecompound of Formula I is R1 is COOH or COOR2.
 4. A method of claim 1wherein the compound of Formula I R1 is
 5. A method of claim 2 whereinthe compound of Formula I R1 is
 6. A method of suppressing the growth ofsebaceous organs in mammals which comprises administering theretobetween 100 and 1000 Mu g/sq. cm/day of: